Truncated EGF receptor

ABSTRACT

The present invention relates to truncated EGF receptor molecules that exhibit increased binding affinities for EGFR ligands such as EGF and TGF-α. The present invention also relates to methods of screening for EGF receptor ligands and methods of treatment which involve the use of these molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Stage Application of PCT InternationalApplication No. PCT/AU00/00782, filed on Jun. 28, 2001 which claimspriority to Australian Patent Application No. PQ8418, filed on Jun. 28,2000, both of which are incorporated herein, by reference, in theirentirety.

FIELD OF THE INVENTION

The present invention relates to truncated EGF receptor molecules and topharmaceutical compositions comprising these molecules. The presentinvention also relates to methods of screening for EGF receptor ligandsand methods of treatment which involve the use of these molecules.

BACKGROUND OF THE INVENTION

The epidermal growth factor receptor (EGFR) family consists of fourdistinct tyrosine kinase receptors, EGFR/HER/ErbB1, HER2/Neu/ErbB2,HER3/ErbB3 and HER4/ErbB4 (1). These receptors are widely expressed inepithelial, mesenchymal and neuronal tissues and play fundamental rolesduring development and differentiation. They are activated by a familyof at least twelve ligands that induce either homo- orhetero-dimerisation of the EGFR homologues. ErbB2 is unable to bindligand on its own but is a potent co-receptor for all ligands whenco-expressed with other members of the EGFR/HER/ErbB family.

The EGFR is a large (1,186 residues), monomeric glycoprotein with asingle transmembrane region and a cytoplasmic tyrosine kinase domainflanked by noncatalytic regulatory regions. Sequence analyses have shownthat the ectodomain (residues 1-621) contains four sub-domains, heretermed L1, CR1, L2 and CR2, where L and CR are acronyms for large andCys-rich respectively (2, 3). The L1 and L2 domains have also beenreferred to as domains I and III, respectively (4). The CR domains havebeen previously referred to as domains II and IV (4), or as S1.1-S1.3and S2.1-S2.3 where S is an abbreviation for small (2).

Many cancer cells express constitutively active EGFR (5) or other EGFRfamily members (6). Elevated levels of activated EGFR occur in bladder,breast, lung and brain tumours. Antibodies to EGFR can inhibit ligandactivation of EGFR and the growth of many epithelial cell lines.Patients receiving repeated doses of a humanised chimeric anti-EGFRmonoclonal antibody (Mab) showed signs of disease stabilization. Thelarge doses required and the cost of production of humanised Mab islikely to limit the application of this type of therapy. These findingsindicate that the development of EGF receptor antagonists may beattractive anticancer agents.

SUMMARY OF THE INVENTION

The present inventors have now made the surprising finding that thedeletion of residues in the CR2 domain of the EGFR ectodomain gives riseto a truncated ectodomain with enhanced affinity for epidermal growthfactors such as (EGF) and/or transforming growth factor-α (TGF-α). Thisfinding goes against recently reported results (8) showing thatdeletions or mutations in the CR2 region reduce EGFR binding affinityfor EGF.

As will be appreciated by those skilled in the art, the truncated EGFRectodomains of the present invention may provide increased sensitivityin assays which screen for ligands of the EGF receptor. Furthermore, thetruncated EGFR ectodomains of the present invention may have therapeuticpotential given their high affinity for ligand and their ability tocompetitively inhibit EGF-induced proliferation responses in vitro.

Accordingly, in a first aspect the present invention provides atruncated EGFR ectodomain, the truncated EGFR ectodomain lacking asubstantial portion of the CR2 domain such that the truncated EGFRectodomain has an increased binding affinity for at least one EGFRligand when compared to the full length EGFR ectodomain.

The EGFR ligand may be, for example, amphiregulin, heparin binding EGF,β-cellulin, EGF or TGF-α. In a preferred embodiment of the first aspectthe truncated EGFR ectodomain has an increased binding affinity for EGFand/or TGF-α.

In a further preferred embodiment of the first aspect the truncated EGFRectodomain lacks at least the third to seventh modules of the CR2domain. In a further preferred embodiment, the truncated EGFR ectodomainlacks at least the second to seventh modules of the CR2 domain. Thetruncated EGFR ectodomain may further lack a portion of the first moduleof the CR2 domain.

In a further preferred embodiment, the truncated EGFR ectodomain lacksresidues 514-621. In yet a further preferred embodiment, the truncatedEGFR ectodomain lacks residues 502-621.

Further deletions or mutations may be made to the L1, CR1 and/or L2sub-domains of the truncated EGFR ectodomain of the present invention,provided that these further deletions or mutations do not substantiallyaffect the binding affinity of the truncated EGFR ectodomain.Preferably, however, the truncated EGFR ectodomain of the presentinvention comprises the L1, CR1 and L2 subdomains.

In a further preferred embodiment, the truncated EGFR ectodomaincomprises residues 1-492 of the EGFR ectodomain. More preferably, thetruncated EGFR ectodomain comprises residues 1-501 or residues 1-513 ofthe EGFR ectodomain.

In a further preferred embodiment, the truncated EGFR ectodomain has anaffinity for EGF such that the K_(d) is less than 30 nM, more preferablyless than 25 nM.

In a further preferred embodiment, the truncated EGFR ectodomain has anaffinity for TGF-α such that the K_(d) is less than 45 nM, morepreferably less than 40 nM.

In a second aspect the present invention provides a polynucleotideencoding a truncated EGFR ectodomain of the first aspect.

In a third aspect the present invention provides an expression vectorcomprising a polynucleotide of the second aspect.

In a fourth aspect the present invention provides a host cell comprisingan expression vector of the third aspect.

In a fifth aspect, the present invention provides a method for producinga truncated EGFR ectodomain of the first aspect, the method comprisingculturing a host cell of the fourth aspect under conditions which allowproduction of the truncated EGFR ectodomain and isolating the truncatedEGFR ectodomain.

In a sixth aspect, the present invention provides a pharmaceuticalcomposition comprising a truncated EGFR ectodomain according to thefirst aspect and a pharmaceutically acceptable carrier or diluent.

In a seventh aspect, the present invention provides a method ofscreening a putative compound for the ability to modulate the activityof the EGF receptor, the method comprising exposing the putativecompound to a truncated EGFR ectodomain according to the first aspectand monitoring the activity of the truncated EGFR ectodomain.

In the context of the seventh aspect, a suitable assay procedure mayinvolve a competition binding assay in a microplate format, where theputative compound is tested for its ability to inhibit the binding oflabelled ligand such as IGF-1 or IGF-2 to the truncated EGF receptorectodomain. The label may be a radiolabelled tag such as ¹²⁵I or afluorescent tag such as fluorescein isothiocyanate or a lanthanide ionsuch as europium.

In an eighth aspect the present invention provides a method of treatingor preventing a disease associated with signalling by a molecule of theEGF receptor family in a subject, the method comprising administering tothe subject a truncated EGFR ectodomain according to the first aspect.

In a preferred embodiment of the eighth aspect, the disease associatedwith signalling by a molecule of the EGF receptor family is selectedfrom psoriasis and tumour states comprising but not restricted to cancerof the breast, brain, ovary, cervix, pancreas, lung, head and neck, andmelanoma, rhabdomyosarcoma, mesothelioma and glioblastoma.

The method of the eighth aspect may be used alone or in combination withother therapeutic measures. For example, the method of the fourth aspectmay be used in combination with cytotoxic modalities, such as anti-EGFRantibodies, radiotherapy or chemotherapy, in the treatment of tumourstates.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: BIAcore analysis of the interactions between sEGFR501 andsEGFR621 with immobilised hEGF or hTGF-α. (A): sEGFR501 (140, 120, 100,80, 60 and 40 nM) was passed over immobilised hEGF (160 RU immobilised).Samples (30 μl) were injected at a flow rate of 10 μl/min. (B): sEGFR501was passed over immobilised hTGF-α (132 RU immobilised). Experimentaldetails were as in panel A. (C): sEGFR621 (1000, 900, 800, 700, 600 and500 nM) was passed over immobilised HEGF. (D): EGFR621 (concentrationsas for panel C) was passed over immobilised hTGF-α. The operatingtemperature was 25° C. At the end of the injection phase, dissociationwas monitored with buffer alone flowing over the sensor surface. Thesurface was regenerated between samples using 10 mM HCl. The signalobtained when the sample was passed over a parallel blank channel hasbeen subtracted electronically to give the specific response.

FIG. 2: Scatchard analysis of equilibrium binding data. The dissociationconstant (KD=1/KA) was calculated from the equilibrium binding responseobtained in FIG. 1 by plotting the data in Scatchard format (Req/nCversus Req; see Experimental Procedures). The slope of the linear fit tothe data gives KA. (A): sEGFR501 versus hEGF; (B): sEGFR501 versushTGF-α;. (C): sEGFR621 versus HEGF; (D): sEGFR621 versus hTGFα.

FIG. 3: Inhibition of EGF-stimulated cell mitogenesis by sEGFR501. (A):The stimulation of ³H-thymidine incorporation by BaF/3ERX cells usingserial dilutions of mEGF. The data was fitted by a sigmoidal function(−) to determine the EC₅₀. (B): Inhibition of the mitogenic response ofBaF/3ERX cells stimulated with mEGF (207 pM) by: sEGFR501 (▪—▪),sEGFR621 (●—●) or anti-EGFR antibody Mab528 (▴—▴). Each point wasassayed in triplicate. Error bars are shown.

FIG. 4: Covalent cross-linking of sEGFR501 dimers after incubation withmEGF. sEGFR501 (5 μM was incubated with (+) or without (−) mEGF (20 μMin 20 mM HEPES (pH 7.4) containing 150 mM NaCl for 1 h at roomtemperature followed by the addition of bis(sulfosuccinimidyl)suberate(BS3, Pierce, Rockford, Ill., USA) to a final concentration of 0.5 mMand incubation for a further 30 min. The reaction was terminated and thedegree of dimer formation was monitored by SDS-PAGE and immunoblottingwith anti-EGFR Mab528 (7) (0.5 μg/ml) andhorseradish-peroxidase-labelled goat anti-mouse IgG (Bio-Rad) withdetection by ECL (Amersham Pharmacia Biotech). Analysis by non-reducingSDS-PAGE was necessary since the antibody used to detect sEGFR501(Mab528) is conformation-dependent.

FIG. 5. Analysis of EGF/sEGFR501 interactions using the analyticalultracentrifuge. (A) Sedimentation equilibrium analysis of EGF, sEGFR501and a mixture of EGF and sEGFR501. The equilibrium distributions wereobtained after centrifugation at 12,000 rpm at 20° C. for 16 h. (□) 20μM EGF; (∘) 10 μM sEGFR501, (Δ) 20 μM EGF+10 μM sEGFR501. The lines ofbest fit drawn though the data for EGF and sEGER501 are for singlespecies and for molecular weight values of 6,000 and 65,600respectively. The line drawn through the data for the EGF/sEGER501mixture is the line of best-fit calculated assuming two species with themolecular weight of the first species fixed at 6,000 and a fitted valueof 106,400 for the molecular weight of the second species. Inset: Theresidual plot for the fit of the EGF/sEGFR501 mixture. (B) Meniscusdepletion sedimentation analysis of the stoichiometry of EGF binding tosEGFR501. Solutions containing 5 μM sEGFR501 and different molar ratiosof EGF:EGFR were spun for 16 h at 20,000 rpm and 20° C. in the XLAanalytical ultracentrifuge. Under these conditions sEGER501 and itscomplexes with EGF are depleted from the meniscus leaving unbound EGF inthe supernatant. Optical density measurements at 280 nm enable theamount of unbound EGF near the meniscus to be estimated. (C) Dataobtained for the weight-average molecular weight of the “second” speciescalculated for mixtures of sEGFR501 (5 μM) and EGF at the concentrationsindicated under the conditions of panel A above. The solid linecorresponds to a simulated curve based on a KD of 30 nM and adimerisation constant of 4 μM.

FIG. 6: BIAcore analysis of the binding of the Gly441Lys sEGFR501 mutantto immobilised hEGF and hTGF-α. Purified Gly441LyssEGFR501 (24-385 nM)was passed over immobilised hTGF-α (Panel A) or hEGF (Panel B) using theexperimental conditions described in FIG. 1. The corresponding Scatchardanalysis, using the equilibrium binding values obtained from thesesensorgrams, is shown below (Panels C,D).

DETAILED DESCRIPTION OF THE INVENTION

When used herein the term “EGFR” is intended to encompass members of theEGF receptor family such as the EGF receptor, ErbB2, ErbB3 and ErbB4. Ingeneral, EGF receptor family molecules show similar domain arrangementsand share significant sequence identity, preferably at least 40%identity.

When used herein, the phrase “full length EGFR ectodomain” refers to theectodomain consisting of residues 1-621 of the EGF receptor. The aminoacid sequence of the full length ectodomain has been previouslydescribed (13). The full length ectodomain contains four sub-domains,referred to as L1, CR1, L2 and CR2, where L and CR are acronyms forlarge and cys-rich respectively.

The CR2 sub-domain consists of the following seven modules joined bylinkers of 2 or 3 amino acid residues and bounded by cysteine residuesas follows:

-   First module: cys residues 482-499-   Second module: cys residues 502-511-   Third module: cys residues 515-531-   Fourth module: cys residues 534-555-   Fifth module: cys residues 558-567-   Sixth module: cys residues 571-593-   Seventh module: cys residues 596-612.-   The results presented herein show that deletions in the CR2 region    of the EGFR ectodomain unexpectedly increase binding affinity of the    ectodomain for EGF and/or TGF-α. In light of this information, a    person skilled in the art would be able to readily generate a number    of candidate truncated ectodomains and screen these candidates for    increased ligand affinity and for therapeutic potential.

For example, truncated ectodomains may be prepared by recombinant DNAtechnology as described herein or as described previously (8).Alternatively, truncated ectodomains may be prepared by subjecting thefull length ectodomain or full length receptor to limited proteolysis asdescribed previously (9).

Binding affinity and inhibitor potency may be measured for candidatetruncated ectodomains using biosensor technology.

Truncated EGFR ectodomains of the invention may be in a substantiallyisolated form. It will be understood that the protein may be mixed withcarriers or diluents which will not interfere with the intended purposeof the protein and still be regarded as substantially isolated. Atruncated ectodomain of the invention may also be in a substantiallypurified form, in which case it will generally comprise the protein in apreparation in which more than 90%, e.g. 95%, 98% or 99% of the proteinin the preparation is a protein of the invention.

In the context of the present invention, the amino acid sequence of thetruncated EGFR ectodomain may be modified provided that the modificationdoes not adversely affect the binding affinity of the truncatedectodomain for at least one EGFR ligand. For example, modifiedectodomains may be constructed by making various substitutions ofresidues or sequences or deleting terminal or internal residues orsequences not needed for binding activity. Generally, substitutionsshould be made conservatively; i.e., the most preferred substitute aminoacids are those having physiochemical characteristics resembling thoseof the residue to be replaced. Similarly, when a deletion or insertionstrategy is adopted, the potential effect of the deletion or insertionon biological activity should be considered. In order to preserve thebiological activity of the truncated ectodomains, deletions andsubstitutions will preferably result in homologous or conservativelysubstituted sequences, meaning that a given residue is replaced by abiologically similar residue. Examples of conservative substitutionsinclude substitution of one aliphatic residue for another, such as Ile,Val, Leu, Met or Ala for one another, or substitutions of one polarresidue for another, such as between Lys and Arg; Glu and Asp; or Glnand Asn. Other such conservative substitutions, for example,substitutions of entire regions having similar hydrophobicitycharacteristics, are well known. Moreover, particular amino aciddifferences between human, murine and other mammalian EGFRs issuggestive of additional conservative substitutions that may be madewithout altering the essential biological characteristics of thetruncated EGFR ectodomains.

Modifications encompassed by the present invention also include variousstructural forms of the primary protein which retain binding affinities.Due to the presence of ionizable amino and carboxyl groups, for example,a truncated ectodomain may be in the form of acidic or basic salts, ormay be in neutral form. Individual amino acid residues may also bemodified by oxidation or reduction.

The primary amino acid structure may be modified by forming covalent oraggregative conjugates with other chemical moieties, such as glycosylgroups, lipids, phosphate, acetyl groups and the like, or by creatingamino acid sequence mutants. Other modifications within the scope ofthis invention include covalent or aggregative conjugates of thetruncated ectodomain with other proteins or polypeptides, such as bysynthesis in recombinant culture as N-terminal or C-terminal fusions.For example, the conjugated polypeptide may be a signal (or leader)polypeptide sequence at the N-terminal region of the protein whichco-translationally or post-translationally directs transfer of theprotein from its site of synthesis to its site of function inside oroutside of the cell membrane or wall (e.g., the yeast α-factor leader).Truncated EGFR ectodomain fusions may comprise peptides added tofacilitate purification or identification of the truncated ectodomain(e.g., poly-His) or to enhance stability or delivery of the ectodomainin vivo.

The truncated EGFR ectodomains of the present invention may also befused to the constant domain of an immunoglobulin molecule. For example,a recombinant chimeric antibody molecule may be produced havingtruncated EGFR ectodomain sequences substituted for the variable domainsof either or both of the immunoglubulin molecule heavy and light chainsand having unmodified constant region domains. This may result in asingle chimeric antibody molecule having truncated EGFR ectodomainsdisplayed bivalently. Such polyvalent forms of the truncated EGFRectodomain may have enhanced binding affinity for EGFR ligands. Detailsrelating to the construction of such chimeric antibody molecules aredisclosed in WO 89/09622 and EP 315062.

As TGFα exists as a membrane bound form, the truncated ectodomains ofthe present invention may be used to target compounds to cancer cells.Accordingly, truncated EGFR ectodomain fusions may comprise compoundsuseful in the diagnosis or treatment of cancer cells such as drugs,isotopes or toxins.

Truncated EGFR ectodomain derivatives may also be obtained bycross-linking agents, such as M-maleimidobenzoyl succinimide ester andN-hydroxysuccinimide, at cysteine and lysine residues. The truncatedectodomains may also be covalently bound through reactive side groups tovarious insoluble substrates, such as cyanogen bromide-activated,bisoxirane-activated, carbonyldiimidazole-activated or tosyl-activatedagarose structures, or by adsorbing to polyolefin surfaces (with orwithout glutaraldehyde cross-linking). Once bound to a substrate, thetruncated ectodomain may be used to selectively bind (for purpose ofassay or purification) anti-EGFR antibodies or EGF.

It may also be desirable to use derivatives of the ectodomains of theinvention that are conformationally constrained. Conformationalconstraint refers to the stability and preferred conformation of thethree-dimensional shape assumed by a peptide. Conformational constraintsinclude local constraints, involving restricting the conformationalmobility of a single residue in a peptide; regional constraints,involving restricting the conformational mobility of a group ofresidues, which residues may form some secondary structural unit; andglobal constraints, involving the entire peptide structure.

It will be appreciated that the truncated EGFR ectodomains of thepresent invention may be used as immunogens, reagents in receptor-basedimmunoassays, or as binding agents for affinity purification proceduresof EGF or other binding ligands.

Truncated EGFR ectodomains may be tested for their ability to modulatereceptor activity using a cell-based assay incorporating a stablytransfected, EGF-responsive reporter gene (10). The assay addresses theability of EGF to activate the reporter gene in the presence of novelligands.

It offers a rapid (results within 6-8 hours of hormone exposure),high-throughput (assay can be conducted in a 96-well format forautomated counting) analysis using an extremely sensitive detectionsystem (chemiluminescence). Once candidate compounds have beenidentified, their ability to antagonise signal transduction via theEGF-R can be assessed using a number of routine in vitro cellular assayssuch as inhibition of EGF-mediated cell proliferation. Ultimately, theefficiency of truncated EGFR ectodomains as tumour therapeutics may betested in vitro in animals bearing tumour isografts and xenografts asdescribed (11, 12).

Truncated EGFR ectodomains of the invention (and substances identifiedby the assay methods of the invention) may preferably be combined withvarious components to produce compositions of the invention. Preferablythe compositions are combined with a pharmaceutically acceptable carrieror diluent to produce a pharmaceutical composition (which may be forhuman or animal use). Suitable carriers and diluents include isotonicsaline solutions, for example phosphate-buffered saline. Thecompositions may be formulated, for example, for parenteral,intramuscular, intravenous, subcutaneous, intraocular, oral ortransdermal administration. Typically, each protein may be administeredat a dose of from 0.01 to 30 mg/kg body weight, preferably from 0.1 to10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.

The routes of administration and dosages described are intended only asa guide since a skilled practitioner will be able to determine readilythe optimum route of administration and dosage for any particularpatient and condition.

In view of the ability of the truncated EGFR ectodomains of the presentinvention to bind strongly to EGFR ligands, the truncated ectodomainswill be useful in diagnostic assays for EGFR ligands, as well as inraising antibodies to the EGFR for use in diagnosis and therapy. Inaddition, purified truncated EGFR ectodomains may be used directly intherapy to bind or scavenge EGFR ligands, thereby providing means forregulating the activities of these ligands. In particular, truncatedEGFR ectodomains of the present invention may be administered for thepurpose of inhibiting EGF-dependent responses.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed in Australia before thepriority date of each claim of this application.

EXPERIMENTAL DETAILS

Materials and Methods

Construction of plasmids used for the expression of truncated forms ofhEGFR ectodomain. The plasmid pEGFR, used in the construction oftruncated hEGFR cDNAs, comprises nucleotides 167-3970 of hEGFR (13) inthe multiple cloning site of plasmid pUC18. Coding is in the oppositesense to the LacZ α peptide, and the insertion is downstream of the XbaIsite of pUC18. This plasmid was used later in excision of the truncatedconstructs for insertion into the mammalian expression vector pEE14(14).

Construction of pEGFR476. An initial plasmid containing nucleotides167-3150 of HEGFR was constructed by ligation of a XbaI/NsiI fragmentfrom pEGFR and XbaI/PstI-cut pBluescript KS+. From this plasmid, a 4 kbpfragment BbsI/BgIII fragment (containing all of pBluescript KS+ andnucleotides 167-1150 and 2951-3150 of EGFR) and a 528 bp BbsI/PvuIIfragment (nucleotides 1151-1679) were ligated with a 70 bp PCR-derivedPvuII and BgIII fragment, encoding amino acids 474-476 of hEGFR, anenterokinase cleavage site and a c-myc epitope tag to facilitatepurification. The 70 bp PCR cassette was produced using a similarprevious construct (15) as template. A plasmid for mammalian celltransfection, pEGFR476, was constructed from this plasmid by ligation ofa 1.6 kbp XbaI/EcoRV fragment 5 with XbaI/SmaI-cut pEE14.

Construction of pEGFR501 and pEGFR513. In each construction PCR was usedwith three oligonucleotides to produce a fragment of hEGFR cDNA(nucleotides 1121 to 1760 or 1121 to 1797 respectively), followed by asequence encoding an enterokinase cleavage site, a c-myc epitope tag anda termination codon. The upstream primer in PCR corresponded to anarbitrary choice of nucleotides 1121-1140 of hEGFR cDNA, while twooverlapping downstream primers were used to construct additionalsequence adjacent to nucleotide 1760 or 1797 respectively. The PCRproducts were cloned using the pCR-Script vector (Stratagene). In eachcase this allowed an ApaI fragment harbouring the newly constructedsequence beginning at nucleotide 1738 of hEGFR, to be excised forsubsequent insertion into the large ApaI fragment of pEGER (whichincluded the entire pUG18 sequence with hEGFR cDNA to nucleotide 1737),in order to prepare a plasmid encoding a truncated hEGFR with XbaIrestriction sites adjacent to the coding sequence. From thesepUC18-based plasmids the fragments harbouring the truncated hEGFR cDNAswere excised by XbaI digestion, and inserted into plasmid pEE14 at theXbaI site to prepare plasmids pEGFR501 and pEGFR513 respectively formammalian cell transfection.

Mutagenesis. The 1.7 kbp fragment harbouring the truncated hEGFR cDNA ofpEGFR501 was introduced into M13 mp18 (16) for mutagenesis.Oligonucleotide-directed in vitro mutagenesis, using the USB-T7 GenTM invitro mutagenesis kit, was employed to produce three single site mutantsof the truncated human sEGFR501 ectodomain, with residues Glu367, Gly441and Glu472 respectively mutated to Lys to match the correspondingresidues in chicken EGFR (4). Clones incorporating the mutations wereidentified by colony hybridisation (17) using ³²P-labelled mutagenicoligonucleotide as a probe, and the mutations were confirmed by DNAsequence analysis (18). Vehicles for mammalian cell expression weregenerated for each mutant by excising the 1.7 kbp fragment harbouringthe mutated sEGFR501 cDNA from M13 RF-DNA by XbaI digestion andinserting it into plasmid pEE14 (16) at the XbaI site.

Cell Culture, DNA Transfection and Protein Analysis. For transienttransfection assays, human 293T fibroblasts maintained in DMEM plus 10%fetal calf serum (FCS) were transfected with plasmid DNA using FuGENE(Roche Molecular Biochemicals, Sydney, NSW) according to themanufacturer's instructions. Supernatants were harvested 48 h aftertransfection, and cell lysates were prepared in NP-40 lysis buffer. Tocharacterise secreted EGFR mutants, aliquots of supernatant and lysatewere immunoprecipitated with a monoclonal antibody (9E10) to the c-myctag, or with Mab 225 (HB-8508, American Type Culture Collection), aconformationally dependent monoclonal antibody recognising theextracellular domain of the hEGFR (19). Immune complexes were collectedon Protein A-Sepharose beads (Zymed Laboratories, Bioscientific Pty.Ltd., Gymea, NSW), fractionated by SDS polyacrylamide gelelectrophoresis (10% gel) and transferred to nitrocellulose membranes.Truncated hEGFR ectodomains and mutants were identified by probingmembranes with horseradish peroxidase (HRP)-conjugated Mab9E10 (Roche),followed by chemilumiscent detection with Pierce Super Signal substrate.

Stable cell lines expressing sEGFR501 were established in the Lec8mutant cell line from CHOK (7) using glutamine synthetase as aselectable marker (15). Supernatants from methionine sulfoximine(MSX)-resistant cell colonies were screened for secreted receptor bybiosensor analysis (see below) or by dot blotting onto nitrocelluloseand probing with HRP-Mab9E10. A single cell line was selected forcloning by limiting dilution.

Lec8 cells expressing sEGFR501 protein were cultured in a Celligen Plusbioreactor (New Brunswick Scientific, New Jersey, USA) using 70 gFibra-Cell Disks carriers with 1.7 L working volume. Continuousperfusion culture using glutamine-free DMEM/F12 medium supplemented withnon-essential amino acids, nucleosides and 10% FCS was maintained for 6weeks. Selection pressure was maintained with 25 μM MSX for the durationof the fermentation. Perfusion rate was adjusted as required to ensure aresidual glucose level of 1.0-1.5 g/L, with a corresponding lactateconcentration of 2.0-2.3 μL.

Purification of Truncated EGFR Ectodomains. For biosensor and AUCanalyses, conditioned medium containing the sEGFR truncated proteins (4L) was adjusted to pH 8.0 with Tris-HCl (Sigma) containing sodium azide(0.02% (w/v)) (TBSA), and particulates removed by centrifugation priorto recovery of c-myc-tagged protein by affinity purification at 4° C. ona column of monoclonal antibody 9E10 covalently-bound to agarose, usingpeptide elution (15). Eluted protein was further purified by sizeexclusion chromatography on Superdex 200 (HR10/30, Amersham PharmaciaBiotech) at room temperature using TBSA buffer at a flow rate of 0.8ml/min. Protein was detected by absorbance at 280 nm.

BIAcore BindingAssays. Protein-protein interactions were monitored inreal time on an instrumental optical biosensor using surface plasmonresonance detection (BIAcore 2000 or 3000, BIAcore, Uppsala, Sweden).Recombinant hEGF or hTGF-α (Gropep, Adelaide, Australia) were purifiedimmediately prior to immobilisation by micropreparative RP-HPLC using aSMART system (Amersham Pharmacia Biotech) as described previously (20).The proteins were immobilised onto the biosensor surface using aminecoupling chemistry (N-hydroxysuccinimide andN-ethyl-N′-dimethylaminopropyl-carbodiimide) at a flow rate of 4 μl/min.Typically 100-200 RU were immobilised equivalent to 0.1-0.2 ng/mm² (20).Automated targeting of immobilisation levels was achieved using theBIAcore 3.1 control software (21).

Prior to analysis, sEGFR621 (23), sEGFR501 and the sEGFR501 mutantsamples were characterised by micropreparative size exclusionchromatography (Superose 123.2/30, Amersham Pharmacia Biotech) to ensuresize homogeneity (20) and pooled fractions were diluted in BIAcorebuffer (HBS: 10 mM Hepes pH 7.4 containing 3.4 mM EDTA, 0.15 mM NaCl and0.005% (v/v) Tween 20) to the appropriate concentration. Typically,samples (30 μl) at concentrations of 10-1000 nM were injectedsequentially over the sensor surfaces at a flow rate of 5 or 10 μl/min.Following completion of the injection phase, dissociation was monitoredin BIAcore buffer at the same flow rate. The sensor surface and sampleblocks were maintained at 25° C. Bound receptor was eluted, and thesurface regenerated between injections, using 40 μl of 10 mM HCl. Thistreatment did not denature hEGF or hTGF-α immobilised onto the sensorsurface, as shown by equivalent signals on re-injection of receptor.

Kinetic rate constants (ka, kd) were determined using the BIAevaluation3.02 software (BIAcore, http//www.biacore.com/products/eval3.html) asdescribed previously (22), or by global analysis using CLAMP (23, 24).Equilibrium binding constants (KA, KD) were determined by directnon-linear least squares analysis of the binding data using an equationdefining steady state equilibrium (KA*Conc*Rmax/(KA*Conc*n);BIAevaluation 3.1). The data was also plotted in Scatchard format(Req/nC versus Req, where Req is the biosensor response at equilibrium,n is the valency and C is the concentration) (25).

Analytical Ultracentrifugation. Experiments were performed using aBeckman XL-A analytical ultracentrifuge (Beckman Coulter, Inc.,Fullerton, Calif.) equipped with absorption optics, using an An60-Tirotor with cells containing quartz windows, as described previously(23). Centrifugation experiments were conducted at 20° C. using a samplevolume of 100 μl. Equilibrium sedimentation distributions obtained at12,000 and 20,000 rpm, were monitored at 280 or 290 nm and analysedusing the program SEDEQ1B (26). The partial specific volume of EGF wastaken as 0.71 ml/g (23).

Chemical Cross Linking. Chemically cross-linked sEGFR501 dimers weregenerated by the incubation of sEGFR501 (5 μm) with mEGF (20 μM in 20 mMHEPES pH7.4 containing 150 mM NaCl for 1 h at room temperature followedby the addition of bis(sulfosuccinimidyl)suberate (BS3, Pierce,Rockford, Ill., USA) to a final concentration of 0.5 mM and incubationfor a further 30 min. The reaction was terminated by the addition ofTris-HCl buffer (pH 7.5) to a final concentration of 10 mM.Monomer-dimer separation was achieved on Novex non-reducing SDS-PAGEgels (10%). Proteins were transferred onto poly(vinyl difluoride) (PVDF)membranes (Bio-Rad, Hercules, Calif., USA) and identified by incubationwith anti-EGFR Mab528 (19) (0.5 μg/ml) followed byhorseradish-peroxidase labelled goat anti-mouse IgG (Bio-Rad) and ECLdetection (Amersham Pharmacia Biotech).

Cell Proliferation Assays. BaF/3ERX cells, a cell line derived fromBaF/3 cells transfected with human EGFR (obtained from Ludwig Institutefor Cancer Research, Melbourne) were washed three times to removeresidual IL-3 and resuspended in RPMI 1640+10% FCS. Cells were seededinto 96 well plates using a Biomek 2000 robotic autosampler (Beckman) at2×10⁴ cells per 200 μl and incubated for 4 h at 37° C. in 10% CO₂.Appropriate concentrations of sEGFR501 or sEGFR621 or the anti-EGFRmonoclonal antibody Mab528, were added to the first titration point andtitrated in two-fold dilutions across the 96 well plate in duplicatewith or without a constant amount of mEGF (207 pM). 3H-Thymidine (0.5μCi/well) was added and the plates were incubated for 20 h at 37° C. in5% CO₂. The cells were then lysed in 0.5 M NaOH at room temperature for30 min before harvesting onto nitrocellulose filter mats using anautomatic harvester (Tomtec, Conn., USA). The mats were dried in amicrowave, placed in a plastic counting bag and scintillant (10 ml) wasadded. ³H-Thymidine incorporation was determined using an automated betacounter (1205 Betaplate, Wallac, Finland).

EXAMPLE 1 Production and Purification of Truncated EGFR Ectodomains

Preliminary analysis of conditioned media from cells transientlyexpressing sEGFR476, sEGFR501 and sEGFR513 showed that only the lattertwo truncated receptors gave detectable binding to hEGF immobilised onthe BIAcore biosensor (data not shown). Stably transfected Lec8 cellsexpressing sEGFR501, were generated and used to produce truncatedreceptor protein at a yield of ˜1.8 mg/L of fermentation medium forphysical-chemical characterisation.

sEGFR501 purified from a Mab9E10 anti-c-myc peptide affinity columnusing peptide elution showed a single symmetrical peak on size exclusionchromatography (apparent molecular mass of ˜80 kDa) and migrated as asingle band of ˜70 kDa on SDS-PAGE under reducing conditions (notshown). sEGFR501 gave a unique expected sequence, LEEKKVXQGT (13) onN-terminal amino acid sequence analysis, the X at cycle 7 being due tothe presence of a disulphide-bonded cysteine residue at that position.The apparent molecular mass of approximately 70 kDa on SDS-PAGE is dueto the residual glycosylation reported for the glycosylation defectiveLec 8 cells (33) since the calculated mass of human sEGFR501 apo-proteinis ˜57.5 kDa. There are eight potential N-linked glycosylation sites insEGFR501 (13) and incubation of sEGER501 with peptide-N-glycosidase(PNG'ase) at 37° C. resulted in the generation of a major band migratingon SDS-PAGE with an apparent molecular mass of 57-58 kDa (data notshown). We have shown previously using BIAcore analysis that removal ofcarbohydrate using PNGase does not affect binding of sEGFR621 to theimmobilised ligand, in agreement with the concept that glycosylation isrequired for correct processing but not for biological activity. Allsubsequent experiments were carried out using the ˜70 kDa sEGFR501.

EXAMPLE 2 Affinity Binding of sEGFR501

The BIAcore biosensor was used to determine both the rate andequilibrium binding constants for the interaction between sEGFR501 andhEGF or hTGF-α. Full length ectodomain (sEGFR621) was used as a positivecontrol for the surface reactivity, since this interaction has beenstudied in detail previously (23, 27).

Representative sensorgrams for the interaction between sEGFR501 orsEGFR621 and hEGF or TGF-α are shown in FIG. 1. Visual inspectionrevealed that the curves approached equilibrium over the concentrationranges tested. Additionally, the hTGF-α sensorgrams appeared to showmore rapid, and virtually complete, dissociation. Thermodynamic analysisof the equilibrium binding data in Scatchard format (FIG. 2) indicatedKD values of 30 and 47 nM (correlation coefficient R=0.993 and 0.999respectively) for the interactions between sEGFR501 and immobilised hEGFor hTGF-α and 412 and 961 nM (R=0.997 and 0.999 respectively) for thecorresponding interactions with sEGFR621. The values obtained byScatchard transformation were also confirmed by direct non-linear leastsquares analysis of the binding data (data not shown) using an equationdefining steady state equilibrium (KA*Conc*Rmax/(KA*Conc*n);BIAevaluation 3.1). Using this analysis, KID values of 32 and 46 nM werecalculated for the interaction between sEGFR501 and immobilised hEGF andhTGF-α respectively and 570 and 959 nM for the interaction betweenfull-length ectodomain (sEGFR621) and immobilised hEGF and hTGF-α. Thevalues obtained with sEGFR621 were in good agreement with those reportedpreviously (23), confirming the surface viability.

The individual rate constants were determined from those parts of thecurves where first order kinetics appeared to be operative (27, 28), andthe corresponding dissociation constants calculated (Table 1). Again,there was good agreement between the KD values calculated in this mannerand those obtained from the equilibrium binding data. It is interestingto note that the binding curves obtained with both sEGFR501 and sEGFR621for hTGF-α appeared to be better fitted to a 1:1 model than thecorresponding data for the hEGF surface (as suggested by the virtuallycomplete dissociation).

TABLE 1 Comparative kinetic data for ligand binding by truncated andfull-length EGFR ectodomains. Interaction k_(a) (M⁻¹s⁻¹) × 10⁻⁵ k_(d)(s⁻¹) K_(D) (nM) sEGFR501/EGF 10-17 0.02 13-21 sEGFR501/TGF-α  9.3-10.50.04 35-40 sEGFR621/EGF 2.9-4.8 0.08 180-300 sEGFR621/TGF-α 0.7-1.0 0.08840-1320

EXAMPLE 3 Antagonist Activity of sEGFR501

The observation that sEGFR501 bound EGF with high affinity prompted usto test whether sEGFR501 would act as a competitive inhibitor for themitogenic stimulation of EGFR in a cell-based assay using the BaF/3ERXcell line. This cell line responds to MEGF with an EC50 of approximately30 pM (FIG. 3A). The competition assay (FIG. 3B) used a constantconcentration of mEGF (207 pM), which causes maximal stimulation (FIG.3A), and varying levels (0.00045-0.5 μM) of sEGFR501, sEGFR621 or theneutralising anti-EGFR monoclonal antibody Mab528 raised againstepidermal growth factor receptors on a human epidermoid carcinoma cellline, A431 (19). This antibody has been shown to prevent the growth ofA431 cell xenografts, bearing high numbers of EGF receptors, in nudemice. The sEGFR501 (IC50=0.02 μM) was almost 10 fold more potent thanthe full-length ectodomain (IC50=0.15 μM) and approximately 3-fold morepotent than the Mab528 anti-EGFR monoclonal antibody (IC50=0.06 μM).

EXAMPLE 4 Dimerisalion of sEGFR501

Chemical cross-linking revealed that sEGFR501 formed dimeric complexesin the presence of ligand. In the presence of 20 μM mEGF, a single highmolecular weight species (apparent Mr 180,000 Da) was formed afterchemical cross-linking which was not detectable when the cross-linkingwas attempted in the absence of ligand (FIG. 4). Western blotting wasemployed to confirm the authenticity of the bands observed, but similardata were obtained with silver or Coomassie blue staining. In addition,size exclusion chromatographic analysis of the reaction mixture, using aTSK G2000SW column developed with a mobile phase of PBS at a flow rateof 0.25 ml/min, showed a peak of apparent Mr 158,000 which correspondedto dimer (data not shown). Similar results have previously been obtainedwith sEGFR621.

Analytical ultracentrifugation showed that the EGF binding sites onsEGFR501 were saturated at an equimolar ratio of ligand and receptorleading to the formation of a 2:2 EGF/sEGFR501 complex (FIG. 5). Thedata for 20 μM EGF alone (FIG. 5A) indicate a single solute of molecularweight 5,980 Da, in good agreement with the value calculated from theamino acid composition (6,040 Da). The molecular weight (65,600 Da) andpartial specific volume (0.71 ml/g) determined for 10 μM sEGFR501 alonewas calculated from the sedimentation equilibrium distribution (FIG. 5A)and is based on the known amino acid composition and a calculated valueof 12% (w/w) for the carbohydrate composition.

Sedimentation equilibrium data for a mixture of EGF (20 μM) and sEGFR501(10 μM) was analyzed assuming two species (FIG. 5A). The molecularweight of the first species was fixed at the value obtained for free EGF(6,000 Da) with the molecular weight and weight fraction of the secondspecies used as fitting parameters. Under these conditions the molecularweight of the second species provides a good approximation to theweight-average molecular weight of sEGFR501 and its complexes. Thebest-fit value showed a complex of weight-average MW 106,400 Da, higherthan predicted for a 1:1 complex (71,600 Da) and more consistent withthe formation of a significant proportion of dimeric 2:2 EGF/sEGFR501complex (see below). High-speed meniscus depletion experiments wereperformed to determine the molar ratio required for saturation ofsEGFR501 with EGF (FIG. 5B). A solution of sEGFR501 (5 μM) was titratedwith EGF to determine the molar ratio at which free EGF is detectable atthe meniscus. The results show that this occurs above 5 μM EGF, implyingan equimolar ratio is required for saturation of the EGF binding site(s)on sEGFR501. These data, taken together with the observed weight averagemolecular weight of the EGF/sEGFR501 complex obtained from theequilibrium analysis (FIG. 5A), confirm that the stoichiometry of theEGF/sEGFR501 dimeric species is 2:2 not 2:1.

Sedimentation equilibrium was used for the analysis of data obtained forsEGFR501 (5 μM) in the presence of a range of EGF concentrations (FIG.5C). The weight average molecular weight obtained for the “second”species increases rapidly as the ratio of EGF/sEGFR501 is increased to1:1 and then tends to plateau around approximately 108,000 at ratiosabove 2:1 (FIG. 5C). The data in FIG. 5A could also be fitted assuming amixture of 1:1 and 2:2 complexes with weight fractions of the monomericand dimeric sEGFR501 complexes of 57% and 31% respectively. Similar datawas obtained with sEGFR621 (23).

EXAMPLE 5 sEGFR501-441 Mutant Binding Studies

Biosensor analysis was also used to analyse the binding of thetransiently expressed sEGFR501 mutants to both immobilised hEGF andhTGF-α surfaces. The presence of the mutant proteins in culturesupernatants from transfected cell lines was demonstrated by bothimmunoblotting with the anti-EGFR monoclonal antibody, Mab 528, andbiosensor analysis using Mab 528 immobilised on the surface. Culturesupernatants from all cell lines showed demonstrable binding to the Mabsurface (441>472=wt>367) (data not shown).

In preliminary experiments, the Glu367Lys mutant and the Glu472Lysmutant showed similar binding characteristics to sEGFR501 when passedover the hEGF sensor surface (data not shown). The Gly441Lys mutantshowed much reduced binding, even though the Mab528 surface hadindicated that the Gly441Lys mutant was present at higher concentrationsthan sEGFR501. Interestingly, when the same samples were passed over theparallel hTGF-α sensor surface the Gly441Lys mutant now showed thehighest binding, whilst the binding of the Glu367Lys mutant theGlu472Lys mutant and wild type sEGFR501 were again similar but lower.

For full biosensor analysis the mutant proteins present in theconditioned media from transient transfected 293T fibroblasts wereconcentrated and purified by a combination of affinity purificationusing the 9E10 monoclonal antibody and size exclusion chromatography onSuperdex 200 and Superose 12. The sensorgrams obtained with theimmobilised hEGF and hTGF-α surfaces (160 and 132 RU immobilisedrespectively) are shown in FIGS. 6A,B. As we had observed in thepreliminary experiments, whilst the binding characteristics of theGlu367Lys and Glu472Lys mutants were essentially undistinguishable fromthose of sEGFR501 shown in FIG. 1 (data not shown) the Gly441Lys mutantagain showed preferential binding to the hTGF-α surface (FIGS. 6A,B).Scatchard analysis of the equilibrium binding data (FIGS. 6C,D)indicated that whilst binding to the TGF-α surface was similar to thatobserved with sEGFR501 (KD=77 nM, correlation coefficient R=0.999), thereactivity of the Gly441Lys mutant towards the EGF surface was nowconsiderably reduced (KD=455 nM, R=0.995). Similar values (78 nM and 469nM) were obtained by direct non-linear least squares analysis of thebinding data (data not shown) using the equation defining steady stateequilibrium.

Kinetic analysis of the binding data (Table 2) indicated that theinteraction with the immobilised TGF-α could be described by anassociation rate constant (ka) of 5.2-6.9×10−5M−1s−1 and a dissociationrate constant (kd) of 0.025s−1 giving a KD=kd/ka of 36-44 nM. Thecorresponding interaction with EGF was described by a ka of1.9-2.3×10−5M−1s−1 and a significantly faster kd of 0.103s−1 giving aKD=kd/ka of 442-545 nM, in good agreement with the results observed fromthe thermodynamic analysis.

TABLE 2 Kinetic analysis of the binding of Gly⁴⁴¹Lys sEGFR501 toimmobilised hEGF and hTGF-α. Interaction k_(a) (M⁻¹s⁻¹) × 10⁻⁵ k_(d)(s⁻¹) K_(D) (nM) TGF-α 5.2-6.9 0.025 36-48 EGF 1.9-2.3 0.103 442-545Discussion

The characteristics of a truncated version of the EGFR ectodomain(sEGFR501) that binds hEGF and hTGF-α with high affinity (Table 1, FIGS.1 and 2) are described herein. The KD values of 13-21 nM for hEGFbinding to sEGFR501 are similar to those, (15-30 nM), seen withchemically cross-linked dimers of full-length EGFR ectodomain and are 10to 25-fold higher than the values generally reported for soluble,full-length EGFR ectodomain derived from either A431 tumour cells,transfected Sf9 insect cells or CHO cells.

sEGFR501, which lacks most of CR2, exhibits ligand-induced receptordimerisation (FIGS. 4 and 5) indicating that the regions responsible fordimerisation are unlikely to include CR2. It also confirms that membraneanchoring is not required for the generation of high affinity dimers incontrast to the situation with ErbB2/ErbB3 heterodimers and neuregulin.The ultracentrifugation analyses showed that the binding sites onsEGFR501 were saturated, and the extent of dimerisation began toplateau, at molar ratios greater than of 1:1 (FIG. 5C), even at therelatively low concentration of sEGFR501 of 5 μM (320 μg/ml). Thiscompares favourably with the small angle X-ray scattering data and ourprevious analytical ultracentrifugation analyses that showed thatsEGFR621 dimerisation, induced by EGF or TGF-α binding, reached amaximum when the ratio of EGF/sEGFR was 1:1.

It is envisaged that sEGFR501 will have therapeutic potential given itshigh affinity for ligand and its ability to competitively inhibitEGF-induced proliferation responses in a model cell system (FIG. 3).This inhibition was greater than that achieved in the same assay with aneutralising monoclonal antibody raised against the receptor (Mab528),chimeric forms of which (C225) are currently in clinical trials.

sEGFR501 was also employed to investigate the residue responsible forthe differential binding between hTGF-α and hEGF observed with chickenEGFR (9). These data demonstrate that the Lys442 in chicken EGFR, whichcorresponds to Gly441 in hEGFR, is the residue responsible fordiscriminating between hTGF-α and hEGF binding.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are apparent to those skilled inmolecular biology or related fields are intended to be within the scopeof the invention.

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1. An isolated truncated ErbB1 ectodomain comprising at least residues1-492 of ErbB1 and lacking at least the third to seventh modules of theErbB1 CR2 domain such that the truncated ErbB1 ectodomain has anincreased binding affinity for at least one ErbB1 ligand when comparedto the full length ErbB1 ectodomain.
 2. The truncated ErbB1 ectodomainas claimed in claim 1 wherein the truncated ErbB1 ectodomain has anincreased binding affinity for EGF and/or TGF-α.
 3. The truncated ErbB1ectodomain as claimed in claim 2 wherein the truncated ErbB1 ectodomainlacks at least the second to seventh modules of the CR2 domain.
 4. Thetruncated ErbB1 ectodomain as claimed in claim 2 wherein the truncatedErbB1 ectodomain further lacks a portion of the first module of the CR2domain.
 5. The truncated ErbB1 ectodomain as claimed in claim 2 whereinthe truncated ErbB1 ectodomain lacks residues 514-621.
 6. The truncatedErbB1 ectodomain as claimed in claim 2 wherein the truncated ErbB1ectodomain lacks residues 502-621.
 7. The truncated ErbB1 ectodomain asclaimed in claim 2 wherein the truncated ErbB1 ectodomain has anaffinity for EGF such that the Kd is less than 30 nM.
 8. The truncatedErbB1 ectodomain as claimed in claim 2 wherein the truncated ErbB1ectodomain has an affinity for TGF-α such that the Kd is less than 45nM.
 9. A chimeric or fusion protein comprising a truncated ErbB1ectodomain as claimed in claim
 2. 10. The chimeric or fusion protein asclaimed in claim 9 wherein the truncated ErbB1 ectodomain is conjugatedto an immunoglobulin constant domain.
 11. The truncated ErbB1 ectodomainas claimed in claim 1 wherein the truncated ErbB1 ectodomain lacks atleast the second to seventh modules of the CR2 domain.
 12. The truncatedErbB1 ectodomain as claimed in claim 1 wherein the truncated ErbB1ectodomain further lacks a portion of the first module of the CR2domain.
 13. The truncated ErbB1 ectodomain as claimed in claim 1 whereinthe truncated ErbB1 ectodomain lacks residues 514-621.
 14. The truncatedErbB1 ectodomain as claimed in claim 1 wherein the truncated ErbB1ectodomain lacks residues 502-621.
 15. The truncated ErbB1 ectodomain asclaimed in claim 1 wherein the truncated ErbB1 ectodomain has anaffinity for EGF such that the Kd is less than 30 nM.
 16. The truncatedErbB1 ectodomain as claimed in claim 1 wherein the truncated ErbB1ectodomain has an affinity for TGF-α such that the Kd is less than 45nM.
 17. A chimeric or fusion protein comprising a truncated ErbB1ectodomain as claimed in claim
 1. 18. The chimeric or fusion protein asclaimed in claim 17 wherein the truncated ErbB1 ectodomain is conjugatedto an immunoglobulin constant domain.
 19. A pharmaceutical compositioncomprising the chimeric or fusion construct as claimed in claim 17 and apharmaceutically acceptable carrier or diluent.
 20. A pharmaceuticalcomposition comprising the truncated ErbB1 ectodomain as claimed inclaim 1 and a pharmaceutically acceptable carrier or diluent.
 21. Anisolated truncated ErbB1 ectodomain comprising residues 1-501 orresidues 1-513 of the ErbB1 ectodomain and lacking at least the third toseventh modules of the ErbB1 CR2 domain such that the truncated ErbB1ectodomain has an increased binding affinity for at least one ErbB1ligand when compared to the full length ErbB1 ectodomain.
 22. Anisolated truncated ErbB1 ectodomain consisting of residues 1-501 or1-513 of the ErbB1 ectodomain.
 23. A dimer of the chimeric or fusionprotein of claim 18 or claim 10.